1. Technical Field
This invention relates to enzyme immunoassays using peroxidase conjugates and, in particular, to improvements in the sensitivity and specificity of such immunoassays provided by polyoxyethylene ether detergents, especially Triton.RTM. X-100.
2. Background Art
The use of antibodies labeled with horseradish peroxidase in immunochemical techniques was first described in 1966 [PK Nakane et al., J. Histochem Cytochem., Volume 14(12), pp. 929-31 (1966)]. The enzyme-linked immunosorbent assay [ELISA] technique was described in 1971 [E. Engvall et al., Immunochemistry, Volume 8(9), pp. 871-4 (1971)]. Horseradish peroxidase remains an important enzyme for labeling antibodies and antigens in a variety of immunochemical techniques because of its ability to rapidly react with substrates which generate easily detectable materials and the relative ease with which the enzyme can be coupled to immunochemicals with the retention of its enzymatic activity. Improvements in enzyme immunoassay technology have concentrated on the development of more specific antibodies with higher binding affinities for their antigens, enzymes demonstrating a faster turnover rate of their substrates, and better means to link antibodies and enzymes with the retention of the activities of both partners.
The sensitivity of an immunoassay can be defined by the ratio of the specific signal generated to the background noise of the system. Factors increasing assay noise include non-specific binding of labeled antibody to various components of the assay and the activity of some endogenous component of the assay matrix which reacts with the enzyme substrate to yield a reaction product interfering with the accurate detection of product formed by the labeled antibody-antigen complex. Typically, additives such as detergents, non-reactive proteins, or salts are added to the assay matrix to lower assay noise by reducing non-specific binding of the enzyme-antibody conjugate or other potentially interfering component to the assay matrix.
Non-ionic detergents are most frequently included in buffer matrices of enzyme immunoassays to reduce noise. Qualtiere et al. [J. Immunology, Volume 119(5), pp. 1645-51 (1977)] suggested that, as a class, non-ionic detergents bind water-soluble proteins at only a few high-affinity sites and that this binding does not generally result in a denaturing effect. These investigators observed that, whereas deoxycholate and dodecylsulfate, ionic detergents, interfered with antigen-antibody reactions, Triton.RTM. X-100 (Rohm & Haas Co., Philadelphia, PA), Nonidet.RTM. P-40 (Shell Co., Houston, TX), Tween.RTM. 20 (Atlas Chemical Industries, Inc., Wilmington, DE), Sterox.RTM. SL (Monsanto Co., St. Louis, MO), and Brij.RTM.-35 (Atlas Chemical Industries, Inc., Wilmington, DE), all non-ionic detergents, did not. U.S. Pat. No. 4,454,232 issued to Breglio et al. on June 7, 1982, claims the use of a non-ionic detergent and a surface active agent to reduce the effects of variable serum protein content of samples which leads to inaccurate estriol immunoassay determinations.
Non-ionic detergents have been shown in several reports to increase the activity of horseradish peroxidase. Porstmann et al. [Clin. Chem. Acta., Volume 109, pp. 175-81 (1981); J. Clin. Chem. Clin. Biochem., Volume 19. pp. 435-39 (1981); Z Med. Laboratoriumsdiagn. Volume 21(3), pp. 180-181 (1980)] found that the inclusion of non-ionic detergents such as polyoxyethylene-octylphenol (Triton.RTM. X-100) or -sorbitol ester (Tween.RTM. 20) in the substrate solution increased the activity of horseradish peroxidase.
Various components of whole blood are known to exhibit peroxidase-like activity which can manifest itself in an enzyme immunoassay using a peroxidase labeled immunoreagent as an increased level of background activity. The amount of these components can vary widely between patient samples. For example, the amount of blood collected on a urogenital swab for the detection of Neisseria gonorrhea antigen can vary depending on the menstrual cycle of the female patient, the degree of infection, or the extent of irritation generated during the taking of the sample. For immunoassays using a visual color change of a peroxidase reactive substrate/chromogen system as a marker of infection, a false positive result may be reported due to the presence of blood. With instrumentally evaluated systems, blood may result in a signal exceeding the threshold level of reactivity and again yield a false positive result. Conversely, eliminating the noise due to blood interference in an immunoassay should increase assay sensitivity by increasing the signal to noise ratio.